Mineral and cellular patterning on biomaterial surfaces

ABSTRACT

Disclosed are advantageous methods for patterning and/or mineralizing biomaterial surfaces. The techniques described are particularly useful for generating three-dimensional or contoured bioimplant materials with patterned surfaces or patterned, mineralized surfaces. Also provided are various methods of using the mineralized and/or patterned biomaterials in tissue engineering, such as bone tissue engineering, providing more control over ongoing biological processes, such as mineralization, growth factor release, cellular attachment and tissue growth.

The present application claims priority to second U.S. provisionalapplication Ser. No. 60/167,289, filed Nov. 24, 1999, which claimspriority to first U.S. provisional application Ser. No. 60/125,118,filed Mar. 19, 1999, the entire text and figures of which applicationsare incorporated herein by reference without disclaimer.

The U.S. Government owns rights in the present invention pursuant togrant numbers R01 DE13033 and T32 GM 08353 from the National Institutesof Health.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the diverse fields oflithography, chemistry, biomaterials and tissue engineering. Moreparticularly, it concerns the patterning and/or mineralization ofbiopolymers. These methods provided are particularly suited to thegeneration of surface-modified three-dimensional biomaterials for use incell culture, transplantation and tissue engineering.

2. Description of Related Art

Many biomedical procedures require the provision of healthy tissue tocounteract the disease process or trauma being treated. This work isoften hampered by the tremendous shortage of tissues available fortransplantation and/or grafting. Tissue engineering may ultimatelyprovide alternatives to whole organ or tissue transplantation.

In order to generate engineered tissues, various combinations ofbiomaterials and living cells are currently being investigated. Althoughattention is often focused on the cellular aspects of the engineeringprocess, the design characteristics of the biomaterials also constitutea major challenge in this field.

In recent years, the ability to regenerate tissues and to control theproperties of the regenerated tissue have been investigated by trying tospecifically tune the mechanical or chemical properties of thebiomaterial scaffold (Kim et al., 1997; Kohn et al. 1997). The majorityof this work has involved the incorporation of chemical factors into thematerial during processing, or the tuning of mechanical properties byaltering the constituents of the material.

The foregoing methods have been used in an attempt to utilize chemicalor mechanical signaling to affect changes in the proliferation and/ordifferentiation of cells during tissue regeneration. Despite suchefforts, there remains in the art a need for improved biomaterials,particularly those with a better capacity to support complex tissuegrowth in vitro (in cell culture) and in vivo (upon implantation).

SUMMARY OF THE INVENTION

The present invention overcomes various drawbacks in the art byproviding a range of improved methods, compositions and devices for usein cell culture, cell transplantation and tissue engineering. Themethods, compositions and apparatus of the invention involve patternedand/or mineralized biomaterial surfaces. The techniques and productsprovided are particularly useful for generating three-dimensional orcontoured bioimplant materials with modified surface features and forgenerating biomaterials incorporating bioactive factors and/or cells.The various methods of using the mineralized and/or patternedbiomaterials in tissue engineering, including bone tissue engineeringand vascularization, thus provide more control over the biologicalprocesses.

Unifying aspects of the invention involve the surface modification,functionalization or treatment of biocompatible materials. Suchmodifications, functionalizations or treatment methods are preferablyused to create reactive surfaces that may be further manipulated, e.g.,patterned and/or mineralized. The patterned and/or mineralizedbiocompatible materials have a variety of uses, both in vitro and invivo.

A first general aspect of the present invention concerns the patternedtreatment of polymer biomaterial surfaces using a unique “diffractionlithography” process. Prior lithographic methods of surface patterninghave been limited to flat, two dimensional surfaces, which is asignificant limitation overcome by the methods provided herein. Thepresent invention is thus applicable to surface patterning on complexthree dimensional biomaterials with surface contours.

The development of these aspects of the overall invention isparticularly surprising as it provides patterns of sufficient resolutionto be useful in biological embodiments. Further advantages of theinvention over the methods of the prior art include the readyincorporation of biologically active components into the patternedbiomaterials and the reduced risk of contamination. Other significantfeatures of the invention are the cost-effectiveness and laborsavingnature of the techniques.

A second general aspect of the invention involves the surface treatmentor functionalization of a biocompatible material, preferably a porous,degradable polymer, such as a film or sponge, to spur nucleation andgrowth of an extended mineral layer on the surface. Such treatment canbe controlled to provide a homogeneous surface mineral layer or apatterned mineral layer, such as islands of minerals. Each of suchextended mineral layers allow the growth of continuous bone-like minerallayers, even on inner pore surfaces of polymer scaffolds.

Such extensively mineralized, patterned mineralized and/orhypermineralized polymers of the invention have advantageous uses inbone tissue engineering and regeneration and tissue vascularization. Theformation of extended mineral islands and/or substantially homogeneous,“continuous” mineral layers, particularly those on the inner poresurfaces of three dimensional matrices, is advantageous as it can beachieved simply (a one step incubation), quickly (about five days), atroom temperature, without leading to an appreciable decrease in totalscaffold porosity or pore size, and is amenable to further incorporationof bioactive substances.

The further incorporation of bioactive substances is exemplified by theformation and use of polymers, preferably, biodegradable polymers, thatare both mineralized and provide for the sustained release of bioactivefactors, such as protein growth factors. In these aspects of theinvention, the type of mineral layer may be controlled by altering themolecular weight of the polymer; the composition of the polymer; theprocessing technique (solvent casting, heat pressing, gas foaming) usedto prepare the polymer; the type and/or density of defects on thepolymer surface; and/or by varying the incubation time.

The various improved biomaterials of the invention have advantageoususes in cell and tissue culture and engineering methods, both in vitroand in vivo. By way of example only, the present invention providesbiomaterial methods and compositions with patterned mineral surfaces foruse in patterning bone cell adhesion.

Accordingly, the general methods of the invention are those suitable forthe surface-modification of at least a first biocompatible material ordevice, comprising:

(a) generating a patterned surface on a biocompatible material or deviceby a method comprising irradiating at least a first photosensitivesurface of a biocompatible material or device with pre-patternedelectromagnetic radiation, thereby generating a pattern on at least afirst surface of the biocompatible material or device; and/or

(b) generating an extended mineralized surface on a biocompatiblematerial or device by a method comprising functionalizing at least afirst surface of a biocompatible material or device and contacting thefunctionalized surface with an amount of a mineral-containing solution,thereby generating extended mineralization on at least a first surfaceof the biocompatible material or device.

The irradiation, lithographic or diffractive lithography methodsgenerally comprise generating a patterned surface on a biocompatiblematerial by a method comprising functionalizing at least a firstphotosensitive surface of a biocompatible material by irradiating thephotosensitive surface with an amount of pre-patterned electromagneticradiation effective to generate a patterned biocompatible materialcomprising a pattern on at least a first surface of the biocompatiblematerial. In these methods, the functionalized surface is preferablyfunctionalized to create a plurality of polar oxygen groups at thesurface, generally so that the functionalized surface can be furthermodified, e.g., with minerals, cells or the like.

It will thus be noted that the methods for generating a patternedsurface on a biomaterial or device, comprise “directly” applyingpre-patterned radiation to a photosensitive surface of a biomaterial ordevice. The “direct” application of the pre-patterned radiation is asignificant advantage as it occurs without the intervention of a “mask”,which is a significant drawback in contact lithography. The presentinvention thus provides “mask-less” or “naked” lithography forbiomaterial patterning in which pre-patterned radiation is impingingdirectly onto a photosensitive surface of a biomaterial in the absenceof an intervening mask.

“Electromagnetic radiation”, as used herein, includes all types ofradiation being electromagnetic in origin, i.e., being composed ofperpendicular electric and magnetic fields. The pre-patterned radiationfor use in the invention is preferably constructively and destructivelyinterfering electromagnetic radiation.

The present invention includes the use of all constructively anddestructively interfering radiation, such as constructive anddestructive interference based on amplitude, as well as phase hologramsthat rely on constructive and destructive interference based on phaseonly. One advantage of phase only holograms is that more light getsthrough, and a more complex pattern can be formed. However, the use ofdiffraction gratings to provide constructive and destructiveinterference based on amplitude is advantageous in construction andcost.

The pre-patterned radiation may be constructively and destructivelyinterfering radiation from any effective part of the visible spectrum.Constructively and destructively interfering radiation in the UV,infrared and visible spectra are preferred examples, with UV and visiblespectra being most preferred.

The pre-patterned, constructively and destructively interferingradiation may be generated by impinging monochromatic radiation on adiffractive optical element that converts the monochromatic radiationinto constructively and destructively interfering radiation.

The monochromatic radiation may be generated from any suitable source.For example, one or more lasers or one or more mercury bulbs. Themonochromatic radiation may be first generated from an electromagneticradiation source and then passed through a suitable filter.

A wide range of diffractive optical elements may be used in theinvention. “Diffractive optical element” is a term that includesdiffraction gratings, holograms, and other pattern generators. There isvirtually no limitation to these aspects of the invention as anycomponent of the spectrum can be patterned by any type of opticalelement by varying the design of the optical element. For example, thereis a well defined relationship between the feature spacing in adiffraction pattern, and the spacing of the slits in the diffractionpattern plus the wavelength of the radiation. Thus, the slit widths canbe varied to create any pattern spacing with any wavelength ofradiation.

Therefore, one may use in the invention one or more diffractive lenses,deflector/array generators, hemispherical lenslets, kinoforms,diffraction gratings, fresnel microlenses and/or a phase-only holograms.Those of ordinary skill in the art will understand that a “diffractiongrating” actually produces an “interference pattern”, not a “diffractionpattern”, which is a matter of semantics resulting from the originalnaming of “diffraction gratings”.

The diffractive optical element(s) may also be fabricated from anysuitable material, such as a transparent polymer or glass. Examples oftransparent polymers are those selected from the group consisting of apoly(methyl methacrylate), poly(styrene), and a high densitypoly(ethylene). Examples of diffraction gratings are those fabricatedfrom metal on glass, metal on polymer or metal with transmissionapertures (slits or holes). Other suitable diffractive optical elementsare those fabricated from fused silica or sapphire. The choice ofelement and matching of element to processing conditions will be routineto those of skill in the art.

Those of ordinary skill in the art will understand that UV light is lesssuitable for use with cells. When using visible light, no compromise ofcell function is expected. Solely as a precaution, an upper limit may beabout 6 W/cm² (Watts per square centimeter). For infrared light, aprecautionary upper limit may be about 2.2 MW/cm² (Megawatts per squarecentimeter).

For use with proteins, a precautionary upper limit of UV may be about 8mW/cm² (Milliwatts per square centimeter). It is not believed that anupper limit of intensity of visible light limits the application of thepresent invention to use with proteins. For use with proteins and cells,local heating during polymerization can be readily minimized, e.g., byusing high molecular weight resins, and by decreasing totalpolymerization time.

Generating a pattern with pre-patterned electromagnetic radiationincludes the direct generation of a patterned surface that naturallyoccurs as a result of the electromagnetic radiation contacting thesurface of the biocompatible material. Therefore, the “photosensitivesurface” of the biocompatible material may simply be the “unmodified”biocompatible material surface. The “thereby generating” of the methodcan therefore be an inherent feature of the method.

“Thereby generating” may also include methods where the irradiatedphotosensitive surface is “developed” to provide the patterned surface.Where the photosensitive surface has not been coated with any particularphotosensitive material, the generation of the patterned surface afterirradiation preferably includes “developing” the irradiatedphotosensitive biomaterial to generate the patterned surface.“Developing” in this sense preferably involves washing or rinsing in asuitable liquid or solvent, such as water or an organic solvent.

The invention further includes more indirect methods of generating thepatterned surface, i.e., where the photosensitive surface to beirradiated is not the unmodified biomaterial surface. In such methods,the photosensitive surface is prepared by applying a photosensitivecomposition, admixture, combination, coating or layer to at least afirst surface of the biocompatible material.

The photosensitive composition may be applied to at least a firstsurface of the biocompatible material by contacting the biocompatiblematerial with a formulation of the photosensitive composition in avolatile solvent and evaporating the solvent to coat the photosensitivecomposition onto the at least a first surface. The photosensitivecomposition may also be applied to at least a first surface of thebiocompatible material by contacting the biocompatible material with aformulation of the photosensitive composition in an aqueous or colloidalsolution to adsorb the photosensitive composition onto the at least afirst surface.

The invention thus comprises:

(a) applying a photosensitive layer to at least a first surface of abiomaterial;

(b) creating pre-patterned radiation;

(c) irradiating the photosensitive layer with the pre-patternedradiation to form an irradiated layer; and

(d) developing the irradiated layer to generate a pattern on the atleast a first surface of the biomaterial.

The invention further comprises:

(a) applying a photosensitive layer to at least a first surface of abiomaterial;

(b) obtaining a monochromatic radiation source;

(c) impinging the monochromatic radiation source on an element thatconverts the monochromatic radiation into patterned radiation;

(d) irradiating the photosensitive layer with the patterned radiation toform an irradiated layer; and

(e) developing the irradiated layer to generate a pattern on the atleast a first surface of the biomaterial.

The invention still further comprises:

(a) applying a photosensitive layer to at least a first surface of abiomaterial;

(b) obtaining a monochromatic radiation source;

(c) transmitting the monochromatic radiation source through an elementthat transforms the monochromatic radiation into patterned radiation;

(d) impinging the transmitted patterned radiation onto thephotosensitive layer of the biomaterial to form an irradiated layer; and

(e) developing the irradiated layer to generate a pattern on the atleast a first surface of the biomaterial.

Any one of a wide variety of photosensitive compositions may be used.Such compositions generally comprise a combined effective amount of atleast a first photoinitiator and at least a first polymerizablecomponent.

Suitable photosensitive compositions may comprise apolymerization-initiating amount of at least a first UV-excitablephotoinitiator, such as a UV-excitable photoinitiator selected from thegroup consisting of a benzoin derivative, benzil ketal,hydroxyalkylphenone, alpha-amino ketone, acylphosphine oxide,benzophenone derivative and a thioxanthone derivative.

Other photosensitive compositions may comprise apolymerization-initiating amount of at least a first visiblelight-excitable photoinitiator, such as a visible light-excitablephotoinitiator selected from the group consisting of eosin, methyleneblue, rose bengal, dialkylphenacylsulfonium butyltriphenylborate, afluorinated diaryltitanocene, a cyanine, a cyanine borate, aketocoumarin and a fluorone dye. These photosensitive compositions mayfurther comprise a co-initiating amount of at least a first co-initiatoror accelerator, such as a co-initiator or accelerator selected from thegroup consisting of a tertiary amine, peroxide, organotin compound,borate salt and an imidazole.

The choice of components for use in the photosensitive compositions willbe straightforward to those of skill in the art. Essentially anyphotoinitiator or initiator system and any “resin” (types of componentsor monomers to be photopolymerized) can be combined. The choice of resinis therefore wide. For example, a suitable “multifunctional acrylate” isany monomer that can be acrylated.

The resin components are used in photopolymerizable amounts, such asphotopolymerizable amounts of at least a first monomeric, oligomeric orpolymeric polymerizable component. Suitable polymerizable monomersinclude those selected from the group consisting of an unsaturatedfumaric polyester, maleic polyester, styrene, a multifunctional acrylatemonomer, an epoxide and a vinyl ether.

One currently preferred photosensitive composition comprises a combinedeffective amount of an eosin photoinitiator, a poly(ethylene glycol)diacrylate polymerizable component and a triethanolamine accelerator.

The methods of the invention produce patterns with a resolution ofbetween about 1 μM and about 500 μM; of between about 1 μM and about 100μM; of between about 10 μM and about 100 μM; of between about 1 μM andabout 10 μM; and of between about 10 μM and about 20 μM. These arehighly suitable for biomedical embodiments, although substantiallyunsuitable for microelectronic embodiments, as a single cell is in the10 μM to 20 μM range. Patterns with a resolution of about 0.5, 0.75, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 50, 75, 100, 150, 200, 250,300, 350, 400, 450, 500, 550 or about 600 μM or so can be produced andused to advantage.

An advantage of the invention is that the entire processes can becarried out at biocompatible temperatures. For example, a biocompatiblematerial can be maintained on a temperature-controlled support duringirradiation.

The biocompatible materials, either before, during or after patterning,may be contacted with an amount of a mineral-containing solutioneffective to generate some, moderate, or preferably extendedmineralization on at least a first surface of the biocompatiblematerial. Such methods link to the mineralization methods and comprisecontacting with a mineral-containing solution prior.

Preferably, the biocompatible material is contacted with themineral-containing solution during or subsequent to the generation ofthe patterned surface, thereby forming a mineralized biocompatiblematerial comprising a pattern of minerals on least a first surface.Furthermore, at least a first mineral-adherent biological cell may besubsequently bound to the mineralized biocompatible material to form apattern of biological cells on least a first surface of thebiocompatible material.

Both the mineral adherence and/or cell adherence may be carried out byexposure of the biocompatible material and/or mineralized biocompatiblematerial to a population of minerals and/or cells either in vitro or invivo. Sequential or simultaneous exposure may be used.

In the mineralization methods of the invention, one generates anextended mineralized surface on a biocompatible material by a methodcomprising functionalizing at least a first surface of a biocompatiblematerial to create a plurality of polar oxygen groups at afunctionalized surface and contacting the functionalized surface with anamount of a mineral-containing solution effective to generate extendedmineralization on the at least a first surface of the biocompatiblematerial.

The methods may comprise generating the functionalized surface byexposing at least a first surface of the biocompatible material to afunctionalizing pre-treatment prior to contact with themineral-containing solution. Effective functionalizing pre-treatmentsinclude exposure to an effective amount of electromagnetic radiation,such as UV radiation; exposure to an effective amount of electron beam(e-beam) irradiation; and exposure to functionalizing biocompatiblechemicals, such as an effective amount of a NaOH solution.

The methods also comprise one-step methods wherein the functionalizedsurface is generated during the contact with the mineral-containingsolution. Such single step methods for forming a mineralized biomaterialthat comprises an extended mineral coating on a biomaterial surfacecomprise incubating a mineralizable biomaterial with an amount of amineral-containing solution, such as an aqueous mineral solution,effective to generate a functionalized biomaterial surface upon which anextended mineral coating forms during the incubation. These methods arepreferred for use with polymer or copolymer biomaterials, such aspolylactic acid (PLA) polymer, polyglycolic acid (PGA) polymer orpolylactic-co-glycolic acid (PLG) copolymer biomaterials.

Any mineralization method, whether pre-patterned or not, may use amineral-containing solution that comprises calcium, wherein theresultant mineralization or extended mineralization comprises anextended calcium coating. Mineral-containing solutions may also compriseat least a first and second mineral, wherein the resultantmineralization or extended mineralization comprises a mixture of thefirst and second minerals. Mineral-containing solutions may furthercomprise a plurality of distinct minerals, wherein the resultantmineralization or extended mineralization comprises a heterogeneouspolymineralized coating.

The methods are controllable to provide mineralization, extendedmineralization, patterned mineralization, extended patternedmineralization, substantially homogeneous mineral coatings,hypermineralized portions or regions, inner pore surfaces of porousmaterials wherein a mineral or an extended mineral coating is generatedon the inner pore surface, and/or pluralities of discrete mineralislands.

Methods for controlling the surface mineralization of biomaterialpolymers comprise altering the molecular weight, polymer composition,ratio of components within the polymer, fabrication technique or surfaceproperties of the biomaterial polymer prior to executing at least afirst surface mineralization process. The methods allow control of thetype of surface mineralization and the degree of surface mineralization,exemplified by the number or size of mineral islands at the surface ofthe biomaterial polymer.

In one example, the biomaterial polymer is a polylactic-co-glycolic acidcopolymer biomaterial and the ratio of lactide and glycolide componentswithin the copolymer composition is altered. In another example, atleast a first surface property of the polymer composition is altered.Further, controlled surface defects may be provided to the polymercomposition to provide a controlled nucleation of discrete mineralislands at the surface of the biomaterial polymer. The density of suchsurface defects may be altered.

The time period of the surface mineralization process may also bealtered. For example, the time of the surface mineralization process maybe extended until discrete mineral islands at the surface of thebiomaterial polymer expand to form a substantially homogeneous mineralcoating at the surface of the biomaterial polymer.

In all such methods, the mineral-containing solution may be a body fluidor a synthetic medium that mimics a body fluid. The biocompatiblematerial may be contacted with the mineral-containing solution byexposure to a natural or synthetic mineral-containing solution in vitroor to a mineral-containing body fluid in vivo.

Any of the foregoing methods, whether for patterning or mineralizationor both, are suitable for direct use with, or for adaptation for usewith, virtually any biocompatible material or device. For example, thebiocompatible materials may comprise at least a first portion that isbiodegradable, non-biodegradable, 3-dimensional, scaffold-like,substantially 2-dimensional, 2-dimensional or film-like. Thebiocompatible materials may comprise at least a first portion that hasan interconnected or open pore structure,

The biocompatible materials may further comprise at least a firstportion that is fabricated from metal, bioglass, aluminate, biomineral,bioceramic, titanium, biomineral-coated titanium, hydroxyapatite,carbonated hydroxyapatite, calcium carbonate, or from anaturally-occurring or synthetic polymer portion. The polymers may beselected from collagen, alginate, fibrin, matrigel, modified alginate,elastin, chitosan, gelatin, poly(vinyl alcohol), poly(ethylene glycol),pluronic, poly(vinylpyrollidone), hydroxyethyl cellulose, hydroxypropylcellulose, carboxymethyl cellulose, poly(ethylene terephthalate),poly(anhydride), poly(propylene fumarate), a polymer enriched incarboxylic acid groups, polylactic acid (PLA) polymer, polyglycolic acid(PGA) polymer, polylactic-co-glycolic acid (PLG) copolymer and PLGcopolymers having a ratio of about 85 percent lactide to about 15percent glycolide.

The biocompatible materials may further comprise at least a firstportion that is prepared by a process comprising gas foaming andparticulate leaching, optionally wherein at least a first bioactivesubstance is operatively associated with the biocompatible materialduring the gas foaming and particulate leaching process.

The gas foaming and particulate leaching process may comprise the stepsof:

(a) preparing an admixture at least comprising a leachable particulatematerial and particles capable of forming a porous, degradable polymerbiomaterial;

(b) subjecting the admixture to a gas foaming process to create aporous, degradable polymer biomaterial that comprises the leachableparticulate material; and

(c) subjecting the porous, degradable polymer biomaterial to a leachingprocess that removes the leachable particulate material from the porous,degradable polymer biomaterial, thereby creating additional porosity.

The leaching process may comprise contacting the porous, degradablepolymer biomaterial with a mineral-containing leaching material.

The biocompatible materials may further comprise at least a firstportion that is a substantially level surface or a contoured surface. Assuch, the biocompatible material may be fabricated as at least a portionof an implantable device.

The foregoing methods and resultant biocompatible materials and devicesmay further comprise a biologically effective amount of at least a firstbioactive substance, bioactive drug or biological cell, two suchbioactive substances, drugs or biological cells or a plurality of suchbioactive substances, drugs or biological cells.

Patterned biocompatible materials may thus be exposed to at least afirst binding-competent mineral, bioactive substance or biological cell,thereby forming a biocompatible material comprising a mineral, bioactivesubstance or biological cell bound in a pattern to at least a firstsurface thereof. Any resultant patterned mineralized biocompatiblematerials may be exposed to at least a first mineral-adherent cell,thereby forming a mineralized biocompatible material comprising at leasta first cell bound in a pattern to at least a first surface of saidbiocompatible material.

Growth factors and/or adhesion ligands may be used to forming growthfactor- or adhesion ligand-coated biocompatible materials comprising atleast a first growth factor or adhesion ligand bound in a pattern to atleast a first surface of said biocompatible material. Such growthfactor- or adhesion ligand-coated biocompatible material may be exposedto at least a first growth factor- or adhesion ligand-adherent cell,thereby forming a mineralized biocompatible material comprising at leasta first cell bound in a pattern to at least a first surface of saidbiocompatible material.

The bioactive substance(s) include DNA molecules, RNA molecules,antisense nucleic acids, ribozymes, plasmids, expression vectors, viralvectors, recombinant viruses, marker proteins, transcription orelongation factors, cell cycle control proteins, kinases, phosphatases,DNA repair proteins, oncogenes, tumor suppressors, angiogenic proteins,anti-angiogenic proteins, cell surface receptors, accessory signalingmolecules, transport proteins, enzymes, anti-bacterial agents,anti-viral agents, antigens, immunogens, apoptosis-inducing agents,anti-apoptosis agents and cytotoxins.

The bioactive substance(s) further include hormones, neurotransmitters,growth factors, hormone, neurotransmitter or growth factor receptors,interferons, interleukins, chemokines, cytokines, colony stimulatingfactors, chemotactic factors, extracellular matrix components, andadhesion molecules, ligands and peptides; such as growth hormone,parathyroid hormone (PTH), bone morphogenetic protein (BMP),transforming growth factor-α (TGF-α), TGF-β1, TGF-β2, fibroblast growthfactor (FGF), granulocyte/macrophage colony stimulating factor (GMCSF),epidermal growth factor (EGF), platelet derived growth factor (PDGF),insulin-like growth factor (IGF), scatter factor/hepatocyte growthfactor (HGF), fibrin, collagen, fibronectin, vitronectin, hyaluronicacid, an RGD-containing peptide or polypeptide, an angiopoietin andvascular endothelial cell growth factor (VEGF).

The biologic cells include bone progenitor cells, fibroblasts,endothelial cells, endothelial cell precursors, stem cells, macrophages,fibroblasts, vascular cells, osteoblasts, chondroblasts, osteoclasts andrecombinant cells that express exogenous nucleic acid segment(s) thatproduce transcriptional or translated products in the cells.

The biocompatible materials may further comprise a combined biologicallyeffective amount of at least a first bioactive substance and at least afirst biological cell. For example, a combined biologically effectiveamount of at least a first osteotropic growth factor or osteotropicgrowth factor nucleic acid and a cell population comprising boneprogenitor cells; or a combined biologically effective amount of VEGF ora VEGF nucleic acid and a cell population comprising.

The at least a first bioactive substance, drug or biological cell may beincorporated into the biocompatible material prior to, during orsubsequent to the surface-modification process. The incorporation intopatterned surface(s) is an advantage as the bioactive substance, drug orbiological cell is bound in a pattern at the patterned surface. Thebiocompatible material may comprise at least a first mineralizedsurface, wherein a mineral-adherent bioactive substance, drug orbiological cell may be bound to the mineralized surface.

The present invention further covers all surface-modified biocompatiblematerials, kits, structures, devices and implantable biomedical deviceswith at least a first portion made by any of the foregoing methods,process or means and combinations thereof. Such surface-modifiedbiocompatible materials may be used in cell culture, celltransplantation, tissue engineering and/or guided tissue regenerationand in the preparation of one or more medicaments or therapeutic kitsfor use for treating a medical condition in need of celltransplantation, tissue engineering and/or guided tissue regeneration.

Methods of the invention include those for culturing cells, comprisinggrowing a cell population in contact with a surface-modifiedbiocompatible material in accordance with the present invention. Thecell population may be maintained in contact with the surface-modifiedbiocompatible material under conditions and for a period of timeeffective to generate a two or three dimensional tissue-like structure,such as a bone-like tissue or neovascularized or vascularized tissue.

Such methods may be executed in vitro or in vivo. The cultured cells maybe separated from a surface-modified biocompatible material and providedto an animal, or may be provided to an animal whilst still in contactwith the surface-modified biocompatible material.

Further methods include those for transplanting cells into an animal,comprising applying to a tissue site of an animal a biologicallyeffective amount of a cell-biocompatible material composition thatcomprises a cell population in operative association with asurface-modified biocompatible material in accordance with the presentinvention.

Still further methods are those for tissue engineering in an animal,comprising applying to a tissue progenitor site of an animal abiologically effective amount of a biocompatible material compositionthat provides a scaffold for tissue growth and that comprises asurface-modified biocompatible material in accordance with the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. FTIR spectra displaying development of phosphate (*) andcarbonate ({circumflex over ( )}) peaks with increasing incubation timein SBF. Peaks representing Poly(lactic-co-glycolic acid) are alsolabelled (o). Incubation times are given to the right of each spectrum.

FIG. 2. Percent mass increase vs. incubation time of scaffolds incubatedin SBF (o), and (▪) control samples incubated in Tris buffer (pH=7.4).Graph shows a trend of increasing mass of SBF-incubated scaffolds,culminating in a 11 +/−2% mass increase after a 16 day incubation.

FIG. 3. The mass of phosphate present in the scaffolds vs. incubationtime.

FIG. 4. Compressive modulus vs. incubation time for scaffolds incubatedin SBF (o), and control scaffolds incubated in Tris buffer (pH=7.4) ().

FIG. 5. Percent mass increase vs. incubation time for PLG scaffoldsincubated in simulated body fluid (SBF). Values represent mean andstandard deviation (n=3).

FIG. 6. The mass of phosphate present in scaffolds vs. incubation timein SBF. Values represent mean and standard deviation (n=3).

FIG. 7. Cumulative release of vascular endothelial cell growth factor(VEGF) from mineralized (X) and non-mineralized ▪ scaffolds. Valuesrepresent mean and standard deviation (n=5).

FIG. 8A. Stimulatory effect of VEGF release from mineralized ▪ andnon-mineralized (□) scaffolds on human dermal microvascular endothelialcells. Cell counts for each release time interval are given as percentsof the control value (striped column) for that interval. Values that aresignificantly larger than their corresponding control are indicated by*'s. Values represent mean and standard deviation (n=5).

FIG. 8B. Sample dose-response curve demonstrating the mitogenic effectof VEGF on human dermal microvascular endothelial cells. Valuesrepresent mean and standard deviation (n=5).

FIG. 9. Incorporation of VEGF into PLG scaffolds during incubation inSBF (♦) and Tris-HCl buffer (o).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Orthopaedic tissue engineering strategies have often focused on the useof natural or synthetic, degradable materials as scaffolds for celltransplantation (cell-based strategies) (Langer and Vacanti, 1993; Craneet al., 1995; Putnam and Mooney, 1996) or as a means to guideregeneration by native osteogenic cells (conductive strategies) (Minabe,1991). Conductive and cell transplantation strategies have been somewhateffective in bone tissue engineering (Ripamonti, 1992; Ishaug-Riley,1997; Shea et al., “Bone formation from pre-osteoblasts on 3-Dscaffolds,” Submitted). The degree of success of these tissueengineering methods is dependent on the properties of the scaffold.

Basic scaffold design requirements include degradability,biocompatibility, high surface area/volume ratio, osteoconductivity, andmechanical integrity. A biocompatible scaffold material that isdegradable over a controllable time scale into non-toxic degradationproducts may disappear in concert with new tissue formation, leaving anatural tissue replacement. A high surface area/volume ratio allows formass transport between cells within the scaffold and the surroundinghost tissue, and provides space for ingrowth of fibrovascular tissue.

Osteoconductivity is important for binding and migration of transplantedand/or native osteogenic cells. Mechanical integrity is required towithstand cellular contractile forces during tissue development toensure maintenance of the initial shape of the scaffold (Kim and Mooney,1998).

The degradability, biocompatibility, and large surface area/volume ratioof scaffolds can be accomplished by the appropriate choice of syntheticor natural material and processing approach. Poly(lactic acid),poly(glycolic acid), and their copolymers have been used in tissueengineering applications because they undergo controllable hydrolyticdegradation into natural metabolites (Gilding, 1981; Li et al., 1990),and can be processed into highly porous scaffolds by a variety ofmethods (Harris et al., 1998; Lo et al., 1995; Mikos and Thorsen, 1994).

A limitation in engineering of many tissue types, including bone tissue,is the inability to induce rapid vascular ingrowth during tissuedevelopment (Mooney et al., 1997). The viability of transplanted cellsand/or host cells that migrate into the scaffold from the native tissueis dependent on transport of nutrients and waste products between thecells and the host tissue.

Transport is initially due solely to diffusion, and cells more thanseveral hundred microns from blood vessels in the surrounding tissueeither fail to engraft or rapidly die due to oxygen deprivation (Colton,1995). Studies indicate that blood vessels will infiltrate a macroporousscaffold to provide enhanced transport to engineered tissues, but theprocess occurs at a rate of less than 1 mm per day and it typicallytakes one to two wk for complete penetration of vascular tissue intorelatively thin (e.g., 3 mm thick) scaffolds (Mikos et al., 1993; Mooneyet al., 1994).

The present invention provides improved biomaterials for use in tissueengineering. Various of the foregoing drawbacks are overcome by thedevelopments of the invention. Certain materials provided arebiodegradable, porous polymers with homogeneous surface layers ofminerals and mineralized inner pores. Porous polymer materials are alsoprovided that have continuous mineral layers in combination withbioactive factors. Other materials provided are patterned materials, towhich any mineral and/or biological component may be bound in aspatially controlled manner.

The patterned and/or mineralized polymers with bioactive factors areprovided to give more control over, or to augment, the processes oftissue formation and regeneration. For example, growth factors can beused that induce cells to behave in a specific manner (Giannobile,1996). Several factors have been identified that induce chemotaxis,proliferation, differentiation, and matrix synthesis of specific celltypes, any one or more of which may be used in the present invention.

Although certain systems have been proposed for factor delivery (Langer,1990; Whang et al., 1998; Wheeler et al., 1998; Shea et al., 1999;Sheridan et al., J. Cont. Rel., In Press), macroporous tissueengineering matrices are still in need of improvement. The inventorsreasoned that the inclusion of bioactive factors into a scaffold wouldallow a higher level of control over cell function within and adjacentto a scaffold construct, thus addressing specific limitations inconductive and cell-based tissue engineering methods.

Certain aspects of the present invention therefore provide scaffoldsthat combine the degradability, biocompatibility and osteoconductivityof mineralized scaffolds with the tissue inductive properties ofbioactive polypeptides. Patterning provides an additional degree ofcontrol. The invention achieves the growth of bone-like mineral on theinner pore surfaces of a scaffold containing a growth factor withoutcompromising factor bioactivity or scaffold porosity. The growth factoris exemplified by vascular endothelial cell growth factor (VEGF), apotent mitogen for human micro and macrovascular endothelial cells,which does not exhibit mitogenic effects on other cell types (Leung etal., 1989).

The mineral- and VEGF-containing matrices of the present invention areparticularly contemplated for use in inducing neovascularizationconcurrent with the engineering of bone tissue. Enhanced vascular tissueformation during tissue development will lead to enhanced viability ofnative and/or transplanted osteogenic cells within a scaffold, enablingthe engineering of a larger volume of bone tissue.

Other bioactive factors for use in this invention include growth hormone(GH); parathyroid hormone (PTH, including PTH1-34); bone morphogeneticproteins (BMPs), such as BMP-2A, BMP-2B, BMP-3, BMP-4, BMP-5, BMP-6,BMP-7 and BMP-8; transforming growth factor-α (TGF-α), TGF-β1 andTGF-β2; fibroblast growth factor (FGF); granulocyte/macrophage colonystimulating factor (GMCSF); epidermal growth factor (EGF); plateletderived growth factor (PDGF); an insulin-like growth factor (IGF) andleukemia inhibitory factor (LIF).

In fact, virtually any hormone, neurotransmitter, growth factor, growthfactor receptor, interferon, interleukin, chemokine, cytokine, colonystimulating factor and/or chemotactic factor protein or polypeptide maybe employed. Further examples include transcription or elongationfactors, cell cycle control proteins, kinases, phosphatases, DNA repairproteins, oncogenes, tumor suppressors, angiogenic proteins,anti-angiogenic proteins, immune response stimulating proteins, cellsurface receptors, accessory signaling molecules, transport proteins,enzymes, anti-bacterial and/or anti-viral proteins or polypeptides, andthe like, depending on the intended use of the ultimate composition.

The biomaterials of the invention with three-dimensional patternedsurfaces allow location-controlled mineralization and cellulardeposition. The three-dimensional, surface-patterned biomaterials of thepresent invention are “smart biomaterials”, which preferentially bindbiological molecules and cells in specific locations. Theregion-specific surface properties allow control over the location andactivity of cells attaching to the biomaterial, when used both in cellculture and in in vivo implantation.

These aspects of the invention represent an important advance, ascontrol over the “locations” of cell deposition and activity isenvisioned to be at least as important as controlling the“characteristics” of cell activity. In fact, controlling the location ofactive cells on the surface of a biomaterial may prove to be the mostimportant determinant in tissue regeneration. The techniques of thepresent invention are particularly advantageous as they provide theability to control the locations of cell presence and activity on thesurface of a biomaterial on a micron scale.

A. Extended Mineral Formation

Certain aspects of the present invention are processes for altering abiomaterial by growing an extended or homogeneous mineral layer on thesurface. Porous, degradable, polymer biomaterials are preferred for suchprocesses, e.g. polylactic acid (PLA), polyglycolic acid (PGA) andpolylactic-co-glycolic acid (PLGA).

The inventors' rationale behind coating these materials with minerals isthat mineral-like coatings are important for bone growth into a porousmaterial and/or for adhesion to a substrate. The basis for this processlies in the observation that in nature, organisms use variousmacromolecules to control nucleation and growth of mineral phases(Campbell et al., 1996; Lowenstein and Weiner, 1989). Thesemacromolecules usually contain functional groups that are negativelycharged at the crystallization pH (Weiner, 1986). It is hypothesizedthat these groups chelate ionic species present in the surroundingmedia, stimulating crystal nucleation (Campbell et al., 1996).

Observations on natural mineralization processes have not previouslybeen adapted for use in connection with biomaterials or tissueengineering processes. However, the present inventors realized that abiomaterial substrate could be functionalized in the laboratory to allowthe induction of mineral deposition.

The inventors further realized that the presence of an extended orhomogeneous mineral layer on the surface of a biomaterial will aid inthe ability to effectively regenerate bone tissue. Described herein arevarious methods for achieving such extended or homogeneous surfacemineralization. However, patterned (or heterogeneous) surfacemineralization is also contemplated for use in certain embodiments, andmay be advantageously achieved by the patterning techniques disclosedherein.

B. Controlling Locations of Cell Activity

Recent advances in the control of cellular processes have shown theutility of controlling the characteristics of cell activity. However,such work has not addressed the specific control of locations of celladhesion to a biomaterial. The present inventors envision the controlover “locations” of cell activity to be as important as control of thecharacteristics of cell activity.

Specifically, in the area of bone tissue regeneration, a prerequisitefor biomaterials to bond to living bone is the formation of a bone-likemineral layer on the biomaterial in the body. This observation suggestedto the inventors that the presence or absence of the mineral maydetermine whether or not bone cells will adhere to and subsequently acton a biomaterial. Thus, they reasoned that specific control over thelocation of mineral on a biomaterial surface will allow control overlocations of bone cell activity. Patterning of minerals on the surfaceof a biomaterial should therefore have a profound effect on theproperties of regenerated bone tissue.

In order to control the locations of cell adhesion to a threedimensional polymer surface, the present invention further providesseveral new methods of treating biopolymers prior to cell seeding. In asurprisingly simple method, pre-treating only certain regions orsub-sections of biopolymer materials, matrices or scaffolds withmineral-containing aqueous solutions results in localized mineralizationin only those areas in contact with the mineralizing solution. Treatmentof the polymer biomaterials on a micron scale is preferably accomplishedusing one of two different processes: surface photolysis and/or surfaceelectrolysis.

The patterned photolysis and electrolysis methods of the presentinvention are suitable for use with porous, biodegradable polymerscaffolds. The surface manipulation methods of the present invention aresurprising in that the inventors' adaptation of techniques from theelectron beam lithography field has allowed, for the first time,patterning applications on three dimensional biomatrices, rather thanbeing limited to two dimensions. Three dimensional matrices aregenerally more effective for creating a biomaterial scaffold for use intissue regeneration.

The various patterning techniques of the invention therefore provide theability to control locations for bone cells specifically (mineralpatterning), and for all cell types in general (polymer surfacetreatment without mineral formation). The differences in processing forcontrol of bone cells versus control of other cell types are describedherein.

As also described in the Detailed Examples, all processes of the presentinvention are room temperature processes. Therefore, specific bioactivesubstances, drugs and proteins, such as adhesion molecules, cytokines,growth factors and the like, can be incorporated into the patterningprocess and resultant biomaterial. Proteins can also be incorporatedinto the cell culture medium, thus patterning the material surface andcausing attachment of specific cells.

C. Photolysis

Certain methods of the invention for functionalizing the surface abiomaterial to allow mineralization and/or control of cellular locationare based upon radiation processes (with or without patterning). Toachieve a homogeneous mineral layer on a biomaterial surface, radiationprocesses without patterning are used. To achieve control of cellularlocation or mineralization, patterned radiation processes are used.

The treatment of polymer biomaterials with electromagnetic (EM)radiation causes surface degradation via a photolysis reaction. Suitableradiation includes all wavelengths of EM radiation, includingultraviolet, visible, infrared, etc. This form of surface degradation,like that achieved with NaOH, causes an increase in the amount of polaroxygen functional groups on the surface of the material.

Interpreting results from distinct studies on bone bonding polymers (Liet al. 1997), the inventors reasoned that the polar oxygen groups formedwould spur mineral nucleation on the surface of the biomaterial whenplaced in a body fluid. Thus, the inventors realized that the ability topattern three dimensional surface functional groups would result in theability to pattern mineral formation and cell adhesion on the surface ofa biomaterial.

Unfortunately, the EM radiation techniques formerly available were alllimited to applications with two dimensional objects. Conventional“contact” optical lithography techniques are so-limited (to twodimensions) due to the requirement for close contact between a mask orcontact grating and the object to be patterned. Thus, prior to thepresent invention, there was no mechanism for producing surface patternson the type of three dimensional, surface-contoured materials that areof most use in tissue engineering.

Lithographic techniques are based upon passing monochromatic EMradiation through an optical grating to produce radiation patterns on ascreen that is on the opposite side of the grating from the EM radiationsource. The pattern formed can be as simple as equally spaced fringesformed by a grating containing equally spaced slits, or as complicatedas a complex hologram.

The present invention significantly advances the tissue engineering artby providing methods for using EM radiation to pattern three dimensionalbiopolymers. In the inventive methods, the “screen” is the polymerbiomaterial. This system amounts to a “diffraction lithography”approach, but the process differs from conventional “contact” opticallithography in that the grating does not act as a mask for the polymer,so that near contact between the grating and the polymer is notnecessary.

In the present methods, the grating produces a pattern of constructiveand destructive interference on the polymer surface. As the grating isnot required to be in near contact with the biomaterial duringtreatment, this diffraction lithography process can be used to treatmaterials with complex three-dimensional surface contours. This is alsoa surprising application of previous technology in that the techniquenow employed would sacrifice line width when used in previousembodiments, so, absent the inventors' particular insight regardingthree dimensional matrix patterning, there would be no motivation todevelop this methodology. Further, the “contact mask” does not need tobe removed, improving the sterile nature of the biotechnique.

Certain types of three dimensional biomatrices envisioned for patterningusing this invention are microsphere and cylindrical matrices. Althougha motivation for developing the present invention was the inventors'goal to develop a process for three dimensional and contouredpatterning, now that the process has been developed, it is equallysuitable for use with two dimensional polymers.

D. Electrolysis

The treatment of polymer biomaterials with electron beam (e-beam)irradiation can also be used to cause surface degradation via anelectrolysis reaction. Surface degradation effects an increase in theamount of polar oxygen functional groups on the surface of the material,which have the same advantageous qualities described herein for thehydrolysis and photolysis reactions.

Surface electrolysis can be patterned on a polymer surface using ascanning electron microscope with basic e-beam lithography capabilities.As shown in the Detailed Examples, this process can also be used totreat materials with flat surfaces or complex three-dimensional surfacecontours.

E. Chemical Hydrolysis

Other methods for surface-functionalizing a biomaterial to allow mineraldeposition utilize chemical pretreatment to achieve surface hydrolysis,e.g., using a NaOH solution. Surface degradation by this techniquecauses an increase in the amount of polar oxygen functional groups onthe surface of the material. The functionalized surface is thenincubated in a mineral-containing solution. The inventors have used suchfunctionalization techniques to allow the generation of a mineralcoating or “hypermineralization”.

Gao et al (1998) recently reported the surface hydrolysis ofpoly(glycolic acid) meshes in order to increase the seeding density andimprove attachment of vascular smooth muscle cells. Although theirprocedure was also based upon the hydrolysis of PGA in NaOH, the polymerscaffold was then directly progressed to the cell seeding experiments(Gao et al., 1998). The present invention instead exposes thesurface-hydrolyzed biopolymer to a calcium-rich solution to inducesurface mineralization.

F. Combined Chemical Hydrolysis and Mineralization

In an unexpected development of the surface-functionalization methods,the inventors surprising found that effective mineral deposition couldbe achieved on biomaterial surfaces without chemical pre-treatment. Inthese methods, a degree of surface hydrolysis sufficient to allowmineralization occurs by simply soaking the biomaterial in an aqueousmineralizing medium. Although pre-treatment, such as by exposure to aNaOH solution, may still be utilized or even preferred in certainembodiments, the one-step mineralization processes have the advantage ofsimplicity and are preferred in certain embodiments.

The one-step mineralization methods utilize the same type ofmineral-containing aqueous solutions as described above, such as bodyfluids and synthetic media that mimic body fluids. Functionalization isfollowed by mineralization in situ, without external manipulation.Although these methods are suitable for use with a wide range ofbiopolymers, the current preferences are for use in conjunction with PLGcopolymers with ratios in the region of 85:15 PLG copolymers, and withbiomaterial scaffolds prepared by gas-foaming/particulate leachingprocesses. The use of 85:15 PLG copolymer scaffolds prepared bygas-foaming/particulate leaching is particularly preferred.

The use of 85:15 PLG copolymers is advantageous as a decrease in thelactide/glycolide ratio of the copolymer is believed to increase therate of surface hydrolysis. However, prior to the present invention, theuse of 85:15 PLG copolymers was disfavored as the mechanical integrityof the polymer declines with increasing glycolide content. Thisinvention shows that 85:15 PLG copolymers can be used effectively as therapid surface hydrolysis allows sufficient mineral formation to offsetthe potential for decreasing integrity, resulting in a sufficient oreven increased overall strength of the composite.

The successes of the present one step mineralization methods (ExampleV), even without foaming/particulate leaching, are in marked contrast toprevious attempts to grow minerals on polyester surfaces. The earliermethods do not result in growth of continuous bone like mineral layers,even after a 6 day incubation in fluids with 50% higher ionicconcentrations than presently used (Tanahashi et al., 1995). Equally, a15 day incubation in essentially the same media fluid as presently usedfailed to produce continuity of mineral microparticles (Zhang and Ma,1999).

The inventors believe that matrix preparation via gasfoaming/particulate leaching techniques results in more surfacecarboxylic acid groups than matrix preparation by other methods (e.g.,solvent casting/particulate leaching). This greater surfacefunctionalization is proposed to contribute to the more rapid nucleationand growth of apatitic mineral observed during the one-stepmineralization processes. Also, the leaching steps of the gasfoaming/particulate leaching methods typically employ mineral solutions,such as 0.1 M CaCl₂, which may further facilitate Ca²⁺ chelation andmore rapid bone-like mineral nucleation.

The techniques of matrix formation by gas foaming/particulate leaching,with or without additional bioactive agents, are described in thefollowing co-owned applications, each of which are incorporated hereinby reference without disclaimer: U.S. patent application Ser. No.09/402,119, filed Sep. 20, 1999, which claims priority to PCTApplication No. PCT/US98/06188 (WO 98/44027), filed Mar. 31, 1998, whichdesignates the United States and claims priority to U.S. ProvisionalApplication Serial No. 60/042,198, filed Mar. 31, 1997; and U.S.application Ser. No. 09/310,802, filed May 12, 1999, which claimspriority to second provisional application Serial No. 60/109,054, filedNov. 19, 1998 and to first provisional application Serial No.60/085,305, filed May 13, 1998.

The studies in Example IX show that bone-like mineral can beadvantageously formed on the inner pore surfaces of matrices prepared bygas foaming/particulate leaching. PLG scaffolds prepared by a gasfoaming/particulate leaching process were successfully mineralized usinga one step incubation in simulated body fluid (SBF) without anyappreciable decrease in total scaffold porosity.

Using gas foamed/particulate leached PLG scaffolds, a 5 day incubationin SBF is sufficient for continuous growth of bone-like mineral on theinner pore surfaces of the scaffold (Example IX). Quantification ofpercent mass gain and phosphate content suggests that the majority ofmineral growth in these aspects of the invention occurs between day 2and day 4 of incubation. These results are even more advanced over theprevious attempts to produce bone-like minerals on polyester surfaces(Tanahashi et al., 1995; Zhang and Ma, 1999).

As these one-step mineralization processes are effective at roomtemperatures, their use to prepare mineralized or hypermineralizedpolymer scaffolds extends to the preparation of mineralized materialsthat include other bioactive substances. It is demonstrated herein thatsuch processes are not detrimental to the activity of biologicalmolecules, such as growth factors. The time- and labor-saving nature ofthese processes therefore make them ideal for preparing matrices for usein many biological processes, especially to stimulate bone growth, whereminerals and growth factors act in concert. The phase, morphology andconstitution of the deposited mineral can be controlled by varying thepH, ionic concentrations and/or temperatures used in the process.

These mineralization techniques are particularly suitable for use withbiodegradable materials. The ability to obtain a continuous bone-likemineral layer within the pores of a three dimensional, porous,degradable scaffold represents a breakthrough in biomaterial processing.The growth of such a continuous bone-like mineral layer is not onlyimportant to cell seeding, but will also likely increase the mechanicalintegrity of these synthetic constructs via a reinforcement mechanism.

Polymer constructs used for tissue engineering applications aregenerally highly porous and do not have mechanical properties in thesame range as bone. Creating an interconnected mineral coating over theinner pore surfaces of a polymer construct, according to these aspectsof the present invention, is therefore a distinct advantage. Thesemethods allow for the production of a hard and stiff exoskeleton,increasing the modulus of a biomaterial and enhancing its resistance tocellular contractile forces during tissue development.

The mineralized scaffold materials of the present invention, e.g., asproduced in Example V, in fact have a post-treatment compressive moduluslarger than those of other poly(α-hydroxy acid) materials used for bonetissue engineering and larger than PLLA bonded poly(glycolic acid) (PGA)matrices that are adequate for resistance of cellular forces duringsmooth muscle tissue development. The materials of this inventiontherefore exhibit shape memory, an important factor in tissueregeneration. The present invention also provides methods to achieveincreases in compressive moduli without notably decreasing scaffoldporosity or pore size, another long-sought after design advantage thatallows cellular migration and vascular infiltration.

G. Mineralization and Growth Factor Release

In addition to showing successful mineralization using a one step, fiveday incubation, the studies of Example IX also demonstrate the sustainedrelease of a bioactive factor (VEGF) from mineralized PLG scaffolds.Three dimensional, porous scaffolds of the copolymer 85:15poly(lactide-co-glycolide) were fabricated by including the growthfactor into a gas foaming/particulate leaching process. The scaffold wasthen mineralized via incubation in a simulated body fluid.

To summarize, the growth of a bone like mineral film on the inner poresurfaces of the porous scaffold is confirmed by mass increasemeasurements and quantification of phosphate content within scaffolds.Release of ¹²⁵I-labelled VEGF was tracked over a 15 day period todetermine release kinetics from the mineralized scaffolds. Sustainedrelease from the mineralized scaffolds was achieved, and growth of themineral film altered the release kinetics from the scaffolds byattenuating the initial burst effect, and making the release curve morelinear. The VEGF released from the mineralized and non-mineralizedscaffolds was over 70% active for up to 12 days following mineralizationtreatment, and the growth of mineral had little effect on total scaffoldporosity.

In more detail, the mineral presence is shown to slow the release of thegrowth factor from the scaffold, resulting in release of a greateramount of factor for a longer time period (e.g., days 3 through 10).After an initial burst release of 44±2% of the incorporated factor inthe first 36 h, the release profile is sustained from the mineralizedsponges for up to 10 days in SBF (FIG. 7). In contrast, the release fromthe non-mineralized scaffolds shows a relatively large initial burst of64±2% over the first 60 h, followed by sustained release for ˜5 days.

The release of a bioactive factor from a mineralized scaffold is animportant result for tissue engineering, particularly bone tissueengineering, because it combines the osteoconductive qualities of abone-like mineral with the tissue inductive qualities of a bioactivefactor, such as a protein growth factor. VEGF release is specificallyuseful in the induction of vascular tissue ingrowth for tissueengineering. This system could also be used with a variety of otherinductive protein growth factors, easily matched to the cell and tissuetypes intended to be stimulated.

Example IX suggests that the growth of mineral on the surface of porousPLG modulates factor release. There is a clear correlation between theonset of mineral growth and the divergence in the release profiles forsamples incubated in SBF and PBS (control). The former occurs betweenday 2 and day 4 of incubation (FIG. 6), while the latter occurs at the 3day time point (FIG. 7). The net effect of mineral presence isattenuation of the initial burst release from the scaffold, andsustained release of a larger amount of factor for a longer period oftime.

The release modulation effect is also apparent in the bioactivity data(FIG. 8A). Release from the mineralized scaffolds has a significantlygreater effect on cell proliferation than release from thenon-mineralized scaffolds during the factor release interval 8-10 days.Examination of the release profiles (FIG. 7) indicates that themineralized scaffolds release a larger amount of VEGF than thenon-mineralized scaffolds during this period.

Previous controlled release formulations using poly(α-hydroxy acid)materials frequently demonstrate a sizeable initial burst in the first1-5 days of release followed by minimal release at later time points(Cohen et al., 1991; Kwong et al., 1986). Achieving relatively constantrelease over a longer period of time is a substantial goal in polymericdrug delivery. Previous attempts to address the “burst effect” have useddouble-walled polymer microspheres (Pekarek et al., 1994) andmicrospheres encapsulated in microporous membranes (Kreitz et al.,1997). The bone-like mineral in this study achieves a functional effectsimilar to the outer layer in double-walled polymeric drug deliverysystems.

The formation of a mineral layer within the pores of PLG scaffolds doesnot notably impair the ability of released growth factor to stimulateproliferation of human dermal microvascular endothelial cells. Thepossibility of protein denaturation and aggregation upon exposure tomoisture is a concern in the controlled release of proteins from certainpolymer systems (Ishaug-Riley et al., 1998). In this case, the proteinis clearly bioactive for eleven days after mineralization treatment (16days after sample preparation).

The 11 day time scale was chosen for analysis in this study because alarge percentage of transplanted cells fail to engraft and die withinthis time period without the development of a vascular supply to augmentmass transport (Mooney et al., 1997; Ishaug-Riley et al., 1998).Sustained release over this time scale induces increased proliferationof endothelial cells, thus supporting angiogenesis during the initialstages of osseous tissue development in vivo.

The present invention therefore provides a system for the sustainedrelease of bioactive factors, such as polypeptides, growth factors andhormones, from mineralized PLG scaffolds. Themineral-biofactor-scaffolds have particular uses in orthopaedic tissueengineering. The presence of a bone-like mineral is a prerequisite toconduction of osteogenic cells into various porous synthetic constructs(Hench, 1991; Kokubo, 1991), and so the mineral is associated withincreased bioreactivity (LeGeros and Daculzi, 1990). The mineral grownby the methods of the present invention thus provides enhancedosteoconductivity in addition to the inductive (e.g., angiogenic) effectof protein release. The growth of the mineral is accomplished via asurprisingly simple single step, room temperature process which,importantly, does not compromise growth factor bioactivity, or totalscaffold porosity.

The following examples are included to demonstrate certain preferredembodiments of the invention. It will be appreciated by those of skillin the art that the compositions and techniques disclosed in theexamples that follow represent compositions and techniques discovered bythe inventor to function well in the practice of the invention, and thuscan be considered to constitute certain preferred modes for itspractice. However, those of skill in the art should, in light of thepresent disclosure, appreciate that many changes can be made in thespecific embodiments that are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

EXAMPLE I Homogeneous Surface Mineralization

A porous, degradable polymer biomaterial is treated for substantiallyhomogeneous mineralization by either pre-treating to induce surfacehydrolysis and then exposing to a mineralizing solution (Examples IIthrough IV) or by conducting a one-step surface hydrolysis andmineralization process (Example V).

Pre-treatment to produce homogeneous surface hydrolysis may be achievedby either soaking in a NaOH solution (Example II) or by treating withelectromagnetic (EM) radiation (Example III). The treated biomaterial isincubated in a mineral-rich, preferably a calcium-rich, fluid, such as abody fluid or synthetic media that mimics body fluid, to spur nucleationand growth of a homogeneous mineral film on the surface (Example IV).

Functionalization and concomitant mineralization can also be achieved bysimply soaking in mineral-containing aqueous solutions, preferably inbody fluids or synthetic media that mimic body fluids. Preparation ofthe polymer biomaterials using a gas-foaming/particulate leachingprocess is generally preferred for such one step mineralization (ExampleV).

Once mineralized, osteogenic cell precursors are seeded onto thebiomaterial in vitro in a cell culture medium. In vivo, bone cellsattach to the biomaterial when implanted.

EXAMPLE II NaOH Pre-Treatment for Surface Mineralized Films

PLGA films (˜25 μm thickness) were prepared by a pressure castingtechnique. Raw polymer pellets were loaded into a mold and placed in aconvection oven at 200 degrees C. The molds were heated under pressure(˜22 N) for 30 sec. and then cooled to room temperature.

For the creation of surface functional groups by NaOH treatment, thefilms were cleansed and immersed in 1.0 N NaOH solution for varyingtimes, up to 10 minutes to create surface functional groups. Followingimmersion, samples were rinsed 3× in distilled water.

EXAMPLE III UV Pre-Treatment for Surface Mineralized Films

PLGA films (˜25 μm thickness) were prepared by a pressure castingtechnique. Raw polymer pellets were loaded into a mold and placed in aconvection oven at 200 degrees C. The molds were heated under pressure(˜22 N) for 30 sec. and then cooled to room temperature.

For the creation of surface functional groups by UV (ultra violet)treatment, membranes were exposed to up to 8 hrs of surface irradiation.

EXAMPLE IV Surface Mineralization after Pre-Treatment

Membranes treated by either NaOH treatment or UV treatment weresubsequently incubated at 37 degrees C. in 50 ml of a simulatedphysiological fluid (SPF, Na: 142 mM, K: 5 mM, Ca: 2.5 mM, Mg: 1.5 mM,Cl: 148 mM, HCO3: 4.2 mM, HPO4: 1 mM, SO4: 0.5 mm) buffered to pH 7.4.Solutions were replaced every 48 hours to ensure that there weresufficient ions in solution to induce mineral nucleation and growth.Following immersion for periods of 120 to 240 hours, samples were dried.

Fourier transform infrared (FTIR) analysis indicates the presence of asurface amorphous apatite. FTIR spectra of scaffolds treated for 0, 2,6, 10, and 16 days indicate the growth of a carbonated apatite mineralwithin the scaffold (FIG. 1). Equivalent spectra were also produced withthe UV-treated films. The broad band at 3570 cm⁻¹ is indicative of thestretching vibration of hydroxyl ions in absorbed water. The peak at1454 cm⁻¹ is indicative of CO₃ ²⁻ν₃, while the 867 cm⁻¹ represents CO₃²⁻ν₂. The peaks at 1097 cm⁻¹ and 555 cm⁻¹ are indicative ofanti-symmetric stretch (ν₃) and anti-symmetric bending (ν₄) of PO₄ ³—,respectively. The peak at 1382 cm⁻¹ represents a NO₃ band.

The presence of OH⁻, CO₃ ²⁻ and PO₄ ³— all indicate that an apatiticlayer has been formed. Other bands representative of apatites are maskedbecause of the strong absorption of the PLGA.

The major peaks at 1755 cm⁻¹ and 1423 cm⁻¹ represent PLGA, and the peakat 1134 cm⁻¹ indicative of C—O stretch in the ester. The peaks at 756cm⁻¹ and 956 cm⁻¹ are indicative of the amorphous domains of thepolymer.

The scaffolds demonstrated an increase in mass over time, culminating ina 11±2% mass gain at the end of the 16 day incubation (FIG. 2). ANOVA ofpercent mass changes of experimental scaffolds reveal a significantdifference in scaffold mass over time (p<0.05), while ANOVA of percentmass changes of control scaffolds does not show a significant differenceover time (p>0.05). Percent mass changes of experimental samples andcontrol samples were significantly different for each time point beyond8 days (p<0.05).

To confirm that the increase in mass was caused by deposition of anapatitic mineral, the mass of phosphate in the scaffolds was nextanalyzed. Phosphate content within the treated scaffolds also increasedsignificantly with the treatment time (FIG. 3). Comparison of phosphatemasses via ANOVA show a statistically significant increase over time(p<0.05), and the differences in phosphate mass between day 8 and 12(p<0.05) and between day 12 and 14 (p=0.05) were also statisticallysignificant. After a 14 day incubation, estimation of the mass ofmineral on the scaffold using phosphate mass data gives 0.76 mg ofhydroxyapatite, while the measured mass increase of the scaffold is1.02±0.40 mg. The fact that the measured value is larger than theestimated value suggests significant carbonate substitution in themineral crystal.

Growth of the BLM layer significantly increased the compressive modulusof 85:15 PLG scaffolds (FIG. 4) without a significant decrease inscaffold porosity. The compressive modulus increased from 60±20 KPabefore treatment to 320±60 KPa after a 16 day treatment, a 5-foldincrease in modulus. ANOVA of modulus changes of experimental scaffoldsreveal a significant difference in scaffold modulus over time (p<0.05),while ANOVA of control modulus data does not show a significantdifference over time (p>0.05). The differences between moduli ofexperimental scaffolds and moduli of control scaffolds werestatistically significant for treatment times of 10 days or longer(p<0.05). The porosity of the scaffolds did not decrease appreciablyafter incubation in SBF. Untreated scaffolds were 95.6±0.2% porous,while scaffolds incubated in SBF for 16 days were 94.0±0.30% porous(n=3). This agrees with the electron micrographs, which displayed only athin (1-10 μm) mineral coating, and thus no significant change in poresize due to mineral growth.

This example shows the successful use of this room temperature processto yield an apatitic surface layer upon a treated polymer surface. Theimportance of room temperature processing is that attachment ofbiological factors is readily achievable, without concern fordenaturation.

EXAMPLE V One Step Mineralization

One step, room temperature incubation processes can also be used tocause nucleation and growth of mineral layers on polymer surfaces. Thisis achieved by incubating polymer scaffolds in mineral-containingaqueous solutions, such as body fluids and synthetic media that mimicbody fluids. These processes are able to grow bone-like minerals withinpolymer scaffolds in surprisingly simple and inexpensive methods. Theeffectiveness of these methods under room temperature conditions rendersthem conducive to the inclusion of bioactive proteins and othermaterials into the processing mineralization.

A first example of one step mineralization concerns the mineraldeposition on porous poly(lactide-co-glycolide) sponges via incubationin a simulated body fluid. The simple incubation technique was used toobtain nucleation and growth of a continuous carbonated apatite mineralon the interior pore surfaces of a porous, degradable polymer scaffold.

A 3-dimensional, porous scaffold of 85:15 PLG was fabricated by asolvent casting/particulate leaching process and incubated in simulatedbody fluid (SBF; NaCl-141 mM, KCl-4.0 mM, MgSO₄-0.5 mM, MgCl₂-1.0 mM,NaHCO₃-4.2 mM, CaCl₂-2.5 mM, and KH₂PO₄-1.0 mM in deionized H₂O,buffered to pH=7.4 with Trisma-HCl). Fourier transform IR spectroscopyand SEM analyses after different incubation times demonstrated thegrowth of a continuous bone-like apatite layer within pores of thepolymer scaffold.

The majority of the mineral growth occurred between days 8 and 12.Mineral growth into a continuous layer likely occurs from day 12, and iscomplete at or before day 16. The mineral grown, being continuous, isthus similar to that in bones and teeth.

The scaffolds demonstrated an increase in mass over time, with an 11±2%gain after 16 days. The increase in mass is due to deposition of anapatitic material. Quantification of phosphate on the scaffold revealedthe growth and development of the mineral film over time with anincorporation of 0.43 mg of phosphate (equivalent to 0.76 mg ofhydroxyapatite) per scaffold after 14 days in SBF. The measured overallmass increase of the scaffold was 1.02±0.4 mg at 14 days. This suggestscarbonate substitution in the mineral crystal.

The compressive moduli of polymer scaffolds also increased fivefold withformation of a mineral film after a 16 day incubation time, as opposedto control scaffolds. This was achieved without a significant decreasein scaffold porosity. The thin mineral coating is thus functionallyimportant, yet mineralization does not change the pore size.

As shown in the mineralization and growth factor studies of Example IX,85:15 PLG scaffolds prepared by gas foaming/particulate leaching exhibiteven more rapid nucleation and growth of apatitic mineral. The 85:15 PLGscaffolds prepared via solvent casting/particulate leaching showed a3±1% increase in mass after a 6 day incubation in SBF. In comparison,85:15 PLG scaffolds prepared by gas foaming/particulate leaching showeda mass increase of 6±1% after a 4 day incubation in SBF.

The even more rapid nucleation and growth of apatitic mineral on 85:15PLG scaffolds prepared by gas foaming/particulate leaching is believedto be due to the increase in carboxylic acid groups caused by the gasfoaming/particulate leaching process, i.e., the greater surfacefunctionalization. Leaching with 0.1 M CaCl₂ also likely facilitateschelation of Ca²⁺ ions, producing more rapid bone-like mineralnucleation.

EXAMPLE VI Bone Cell Control

Polymer biomaterial is treated to form a patterned biosurface,preferably suing either patterned EM radiation or electron beamirradiation. Treated biomaterial is washed with distilled water toremove residual monomers from the surface photolysis or electrolysis.

The treated biomaterial is incubated in a mineral-rich, preferably acalcium-rich, fluid, such as a body fluid or synthetic media that mimicsbody fluid, to spur nucleation and growth of mineral on the treatedregions of the polymer. This results in a mineral pattern on the surfaceof the polymer. This step can be done either in vitro, using a bodyfluid or simulated body fluid; or in vivo, where the natural body fluidperforms this function.

Osteogenic cell precursors are seeded onto the biomaterial in vitro in acell culture medium. In vivo, bone cells attach to the biomaterial whenimplanted. In either case, cells adhere preferentially to mineralizedportions of the substrate.

EXAMPLE VII Diffraction Lithography

Previous studies on the control of locations of cell adhesion to abiomaterial surface have utilized conventional UV lithography to patterna two dimensional polymer surface (Pierschbacher & Ruoslahti, 1984);Ruoslahti & Pierschbacher, 1987); Matsuda et al., 1990); Britland etal., 1992); Dulcey et al., 1991); Lom et al., 1993); Lopez et al.,1993); Healy et al., 1996).

In the prior techniques, the two-dimensional biomaterial surface iscoated with a thin layer of photoresist (PR), the PR is exposed througha metal mask, and the exposed PR is removed in solvent, leaving a PRmask on the surface of the biomaterial sample. The surface of thepolymer biomaterial is then chemically or physically treated through thePR mask, and the mask is removed by a solvent after treatment.

The former processes requires a flat, two dimensional biomaterial, whichsuffices for studying the effects of surface treatment on cell activity,but is not sufficient for the treatment of typical biomaterials, whichhave three dimensional surface contours.

In the present methods, suitable for use with three dimensionalpolymers, the grating produces a pattern of constructive and destructiveinterference on the polymer surface. As the grating is not required tobe in near contact with the biomaterial during treatment, thisdiffraction lithography process can be used to treat materials withcomplex three-dimensional surface contours. However, the process isequally useful in connection with two dimensional biomaterials.

EXAMPLE VIII Control of Other Cell Types

Polymer biomaterial is treated to form a patterned biosurface,preferably using either patterned EM radiation or electron beamirradiation. Treated biomaterial is washed with distilled water toremove residual monomers from the surface photolysis or electrolysis.

The treated biomaterial is incubated in a solution containing bioactivemolecules or proteins, such as growth factors, adhesion molecules,cytokines and such like, which promote adhesion of a specific cell type.Cells are seeded onto the biomaterial in vitro in a cell culture medium.In vivo, cells attach to the biomaterial when implanted. In either case,cells adhere preferentially to the treated portions of the substrate.

The use of specific agents or proteins, such as growth factors, thatpromote attachment of certain cell types, gives the potential to patternany cell type on the three dimensional surface of the polymer, both invitro and in vivo.

EXAMPLE IX Growth Factor Release from Mineralized Matrices

A. Materials and Methods

1. Gas Foaming-Particulate Leaching

Poly(lactide-co-glycolide) pellets with a lactide:glycolide ratio of85:15 were obtained from Medisorb, Inc. (I.V.=0.78 dl/g) and ground to aparticle size between 106 and 250 μm. Ground PLG particles were thencombined with 250 μl of a 1% alginate (MVM, ProNova; Oslo, Norway)solution in ddH₂O, and 3 μg of vascular endothelial cell growth factor(VEGF, Intergen; Purchase, N.Y.), and vortexed. These solutions werelyophilized, mixed with 100 mg of NaCl particles (250 μm<d<425 μm), andcompression molded at 1500 psi for 1 min in a 4.2 mm diameter die. Thisyielded 2.8 mm thick disks with a diameter of 4.2 mm.

Disks were then exposed to 850 psi CO₂ gas in an isolated pressurevessel and allowed to equilibrate for 20 h. The pressure was decreasedto ambient in 2 min, causing thermodynamic instability, and subsequentformation of gas pores in the polymer particles. The polymer particlesexpand and conglomerate to form a continuous scaffold with entrappedalginate, VEGF, and NaCl particles. After gas foaming, the disks wereincubated in 0.1 M CaCl₂ for 24 h to leach out the salt particles andinduce gellation of the alginate within the polymer matrix. Alginate wasincluded in the scaffolds because it has been shown to abate the releaseof VEGF from PLG scaffolds (Wheeler et al., 1998).

2. Mineralization

Certain scaffolds were mineralized via a 5 day incubation in a simulatedbody fluid (SBF). Simulated body fluid (SBF) was prepared by dissolvingthe following reagents in deionized H₂O: NaCl-141 mM, KCl-4.0 mM,MgSO₄-0.5 mM, MgCl₂-1.0 mM, NaHCO₃-4.2 mM, CaCl₂-2.5 mM, and KH₂PO₄-1.0mM. The resulting SBF was buffered to pH=7.4 with Trisma-HCl and held at37° C. during the incubation periods. The SBF solutions were refresheddaily to ensure adequate ionic concentrations for mineral growth.

The porosity of scaffolds was calculated before and after mineralizationtreatment using the known density of the solid polymer, the knowndensity of carbonated apatite, the measured mass of mineral and polymerin the scaffolds, and the volume of the scaffold.

3. Characterization of Mineral Growth

To analyze mineral growth on gas foamed PLG scaffolds, sets of threescaffolds were incubated in SBF for periods ranging from 0-10 days.Samples were removed from solution and analyzed after 0, 2, 4, 8, and 10day incubation periods. The dry mass of each scaffold was measuredbefore and after incubation in SBF, and percent increases in mass werecalculated and compared using ANOVA and a Student's t-test to revealsignificant differences in mass for different SBF incubation times.

The amount of phosphate present in the scaffolds after theaforementioned incubation times was determined using a previouslydescribed colorimetric assay (Murphy et al., J. Biomed. Mat. Res., InPress; incorporated herein by reference). The phosphate mass data werealso compared using ANOVA and a Student's t-test to reveal significantdifferences in mass for different SBF incubation times.

To estimate the amount of apatite on the scaffold after a 6 dayincubation, the measured mass of phosphate was multiplied by the knownratio of mass of hydroxyapatite [Ca₁₀(PO₄)₆(OH)₂, f.w.=1004.36 g] tomass of phosphate in hydroxyapatite (569.58 g). This is a conservativeestimate, since it assumes that all phosphate is being incorporated intostoichiometric hydroxyapatite. This mineral mass estimate increases ifone assumes increasing substitution of carbonate into the mineralcrystal.

4. VEGF Release Measurements

In order to assess the incorporation efficiency of VEGF into the PLGscaffolds and to track the VEGF release kinetics from the scaffolds,receptor-grade ¹²⁵I-labeled human VEGF (90 μCi/μg; BiomedicalTechnologies Inc.; Stoughton, Mass.) was utilized as a tracer. In placeof the 3 μg VEGF in the normal sample preparation, 0.5 μCi ofradiolabeled VEGF was added to each matrix. To assess VEGF incorporationefficiency, the total incorporated activity was compared to the activityof the initial ¹²⁵I VEGF sample prior to incorporation into thescaffolds.

To determine the effects of mineral growth on factor release, releasekinetics were measured both in SBF during mineral formation and inphosphate buffered saline (PBS). Scaffolds prepared with radiolabeledVEGF were placed in 4 ml of SBF or PBS and held at 37° C. At various settimes, the scaffolds were removed from solution and their radioactivitywas assessed using a gamma counter. After each analysis, solutions wererefreshed and scaffolds were placed back into solution.

The amount of radiolabeled VEGF released from the scaffolds wasdetermined at each time point by comparing the remaining ¹²⁵I VEGF tothe total originally loaded into each scaffold. The percent release ofVEGF from scaffolds incubated in SBF was compared to that of scaffoldsincubated in PBS at each time point via a Student's t-test to revealsignificant differences in cumulative release.

5. Biological Activity of Released VEGF

The biological activity of VEGF incorporated into, and released from,polymer matrices was determined by testing its ability to stimulate thegrowth of cultured human dermal microvascular endothelial cells isolatedfrom neonatal dermis (HMVEC(nd), Cascade Biologics; Portland, Oreg.).

HMVEC(nd) were cultured to passage 7 in MCDB 131 media (CascadeBiologics) supplemented with Cascade Biologics' microvascular growthsupplement (5% fetal bovine serum, hydrocortisone, human fibroblastgrowth factor, heparin, human epidermal growth factor, and dibutyrylcyclic AMP) prior to use. Cells were plated at a density of 5×10³cells/cm² on 12 well tissue culture dishes (Corning; Cambridge, Mass.)which were precoated with 1 μg/cm² human plasma fibronectin (LifeTechnologies, Grand Island, N.Y.). The cells were allowed to attach for24 h, and the media in each well was replaced then with 3 ml ofserum-free media (Cell Systems; Kirkland, Wash.) supplemented with 50μg/ml gentamicin (Life Technologies).

A 12 mm transwell (3 μm pore diameter, Corning) containing eithermineralized, or non-mineralized, VEGF releasing matrix was placed ineach experimental well (n=5 for each group), while mineralized matricescontaining no VEGF were placed in the control wells (n=5). To determinethe dose response to known concentrations of VEGF, additional wells (n=4per concentration) were supplemented with 40, 20, 10, and 5 ng/ml ofsoluble VEGF which had not been incorporated into matrices.

After 72 h all of the cells in the experimental and control wells wereremoved with a solution of 0.05% trypsin/0.53 mM EDTA (LifeTechnologies), and counted using a ZM Coulter counter (Coulter; Miami,Fla.). The transwells containing the matrices were immediatelytransferred to new fibronectin-coated (1 μg/cm²) wells that had beenseeded with cells (5×10³ cells/cm²) 24 h before, and allowed to incubatefor an additional 72 h before the cells were removed and counted. A newset of VEGF dose response wells were also set up concurrent with thetransfer of the transwells. The 72 h cycles were continued for 12 days.

Cell counts in experimental wells were compared to cell counts incontrol wells for each 72 h interval using a Student's t-test to revealsignificant differences in HMVEC proliferation.

B. Results

1. Mineralization

Incubation of gas foamed 85:15 poly(lactide-co-glycolide) scaffoldscontaining VEGF resulted in the growth of bone-like mineral on the innerpore surfaces. Analysis of variance showed that differences in percentmass gain with SBF incubation time were significant (p<0.05). Thescaffolds showed an increase in mass with incubation time, with a 6±1%mass gain after a 4 day incubation in SBF (FIG. 5). The scaffold masssubsequently remained relatively constant. The increase in mass betweentwo day and four day incubation times was significant p<0.05), whilethere was no significant difference in percent mass gain between thefour day incubation time and the longer incubation times (p>0.05).

To verify that the increase in mass was caused by the deposition of anapatitic mineral, the mass of phosphate in the scaffolds was analyzed.Phosphate content within scaffolds increased with SBF incubation time(FIG. 6). Analysis of variance showed that differences in phosphatecontent with SBF incubation time were significant (p<0.05). Thedifference in phosphate content between the two day and six dayincubation times was significant (p<0.05), while there was nosignificant difference between the phosphate mass of the six dayincubation time and longer incubation times (p>0.05).

The inventors have previously shown that the increase in mass andphosphate content in these scaffolds indicates growth of a continuousbone-like mineral film on the inner pore surfaces (Murphy et al., J.Biomed. Mat. Res., In Press).

The total porosity of the scaffolds after a 10 day incubation in SBF was92±1%, which is similar to the initial scaffold porosity (93±1%).

After a 6 day incubation, estimation of the mass of mineral on thescaffold using phosphate mass data gives 0.10 mg of hydroxyapatite,while the measured mass increase of the scaffold is 0.39±0.03 mg. Thefact that the measured value is larger than the estimated value suggestssignificant carbonate substitution in the mineral crystal.

2. VEGF Release and Activity

Vascular endothelial cell growth factor (VEGF) was incorporated into PLGscaffolds with an efficiency of 44±9% and released over a 15 day periodin SBF and PBS solutions. An initial burst release of the incorporatedgrowth factor was observed over the first 12-36 h followed by asustained release for the remainder of the study (FIG. 7).

The cumulative release from scaffolds incubated in SBF becamesignificantly smaller than release from scaffolds incubated in PBS after3 days, and this difference remained significant through 10 days ofrelease (p<0.05). At time points beyond 10 days there is no significantdifference in cumulative release from scaffolds incubated in SBF versusthose incubated in PBS (p>0.05).

VEGF released from mineralized and non-mineralized scaffolds had amitogenic effect on human dermal microvascular endothelial cells(HMVECs).

Cells were grown in wells containing three different scaffold types: 1)Mineralized, VEGF-containing scaffolds (MV scaffolds); 2)non-mineralized, VEGF containing scaffolds (NV scaffolds); and 3)mineralized control scaffolds without VEGF (MC scaffolds). Cells grownin wells containing MV and NV scaffolds demonstrated significantlyincreased proliferation when compared with cells grown in wellscontaining MC scaffolds (FIG. 8A). Cell counts were significantly higherin wells containing MV and NV scaffolds for all time intervals (p<0.05)with the exception of the wells containing NV scaffolds over the 14-16day factor release interval.

During the 8-10 day factor release interval, MV scaffolds showed asignificantly greater mitogenic effect on HMVECs than NV scaffolds(p<0.05). There was no significant difference in the stimulatory effectof MV scaffolds versus NV scaffolds for any other time interval(p>0.05).

A dose-response curve (FIG. 8B) generated for the HMVECs was used tocalculate an effective concentration for the released growth factor.Comparison of this effective concentration with the amount of VEGF knownto be released during each time interval (FIG. 7) indicates that thereleased VEGF is over 70% active for all time intervals.

EXAMPLE X Effects of Growth Factors on Mineralization

A. Materials and Methods

Poly(lactide-co-glycolide) pellets with a lactide:glycolide ratio of85:15 were obtained from Medisorb, Inc. (I.V.=0.78 dl/g) and ground to aparticle size between 106 and 250 μm. Ground PLG particles were thencombined with 250 μl of a 1% alginate (MVM, ProNova; Oslo, Norway)solution in ddH₂O, and vortexed. These solutions were lyophilized, mixedwith 100 mg of NaCl particles (250 μm<d<425 μm), and compression moldedat 1500 psi for 1 minute in a 4.2 mm diameter die. This yielded 2.8 mmthick disks with a diameter of 5.0 mm.

Disks were then exposed to 850 psi CO₂ gas in an isolated pressurevessel and allowed to equilibrate for 20 hours. The pressure wasdecreased to ambient in 2 minutes, causing thermodynamic instability,and subsequent formation of gas pores in the polymer particles. Thepolymer particles expand and conglomerate to form a continuous scaffoldwith entrapped alginate, and NaCl particles. After gas foaming, thedisks were incubated in 0.1M CaCl₂ for 24 hours to leach out the saltparticles and induce gellation of the alginate within the polymermatrix. Alginate was included in the scaffolds because it has been usedin VEGF release studies to help abate the release of VEGF from PLGscaffolds, and it was necessary to precisely mimic the scaffoldconditions during factor release studies.

The total porosity of scaffolds was calculated using the known densityof the solid polymer, the measured mass polymer in the scaffold, and themeasured volume of the scaffold. Cross sectional electron micrographs ofscaffolds were obtained by bisecting the scaffolds via freeze fractureand imaging using a Hitachi S3200N scanning electron microscope.

To assess the effect of VEGF in solution on the mineral growth process,scaffolds were incubated in SBF containing 0.2 μCi ¹²⁵I VEGF (Receptorgrade human VEGF, 90 μCi/μg, Biomedical Technologies Inc.; Stoughton,Mass.) (n=5). Samples were incubated for five days, since this is thetime period required for growth of a significant amount of bone-likemineral within the inner pore surfaces of gas foamed/particulate leached85:15 PLG scaffolds. After incubation, scaffolds were washed three timesin ddH₂O, and assessed for radioactivity using a gamma counter. Thepercent incorporation of VEGF into the polymer scaffolds was calculated(Counts of scaffold/counts of solution *100) and plotted vs. incubationtime.

The incubation was done in tubes that were siliconized using sigmacote,then presoaked in a 1% bovine serum albumin (BSA) solution for 30minutes to coat the tube surface with BSA and thus reduce binding ofVEGF to the inner surface of the tubes. Solutions were refreshed dailyto ensure sufficient ionic concentrations for mineral growth andconstant concentration of the iodinated growth factor in the solution.

Simulated body fluid (SBF) was prepared daily by dissolving thefollowing reagents in deionized H₂O: NaCl-141 mM, KCl-4.0 mM, MgSO₄-0.5mM, MgCl₂-1.0 mM, NaHCO₃-4.2 mM, CaCl₂-2.5 mM, and KH₂PO₄-1.0 mM. Theresulting SBF was buffered to pH=7.4 with Trisma-HCl and held at 37° C.during the incubation periods.

B. Results

85:15 Poly(lactide-co-glycolide) scaffolds prepared via a gasfoaming/particulate leaching process were 93±1% porous and displayed anopen pore structure with a pore diameter of ˜200 μm.

The incorporation of radioactive VEGF into the scaffolds was larger forcontrol scaffolds than for experimental scaffolds for all time pointsbeyond 2 days (p<0.05). The control data also show a trend of increasingincorporation of VEGF with increasing incubation time (FIG. 9). Thesedata indicate that VEGF is being incorporated into the control scaffoldsmore efficiently than it is being incorporated into the experimentalsamples, and the amount of VEGF in the experimental scaffolds is notincreasing during mineralization treatment.

The data show that VEGF does not significantly incorporate into PLGscaffolds during incubation in SBF. There is also no significantincorporation of the growth factor into the mineral during the initialstages of mineral growth. Thus, the previously shown attenuation of VEGFrelease from PLG scaffolds during mineral growth cannot be explained byincorporation of protein into the mineral film, binding of the proteinto the scaffold surface, or diffusion of the protein back into thescaffold during protein release.

The postulated steps in the mineral growth process on PLG scaffoldsare: 1) surface functionalization via a hydrolysis reaction; 2)Chelation of Ca²⁺ ions by surface carboxylate anions; 3) Nucleation andgrowth of mineral crystals on the polymer surface. The lack ofincorporation of VEGF into PLG scaffolds incubated in SBF indicates thatthe protein does not compete with calcium ions for binding sites on theinner pore surfaces of the scaffolds or efficiently diffuse back intothe scaffolds after release.

In this case, the amount of protein incorporated into the scaffolds wassignificantly larger for control samples incubated in Tris-HCl buffer,and the incorporation increased over time. The increased efficiency ofincorporation of VEGF into control samples may be due to more efficientdiffusion of the factor into control scaffolds, or enhanced binding ofthe factor to the inner pore surfaces of the control scaffolds. Thisresult shows that the effects of mineral growth on VEGF release from PLGscaffolds cannot be explained incorporation of VEGF back into PLGscaffolds after release, or binding of VEGF to the scaffold's inner poresurfaces.

There is no significant incorporation of protein into the mineral filmduring the initial stages of mineral growth. During incubation of PLGscaffolds in SBF containing ¹²⁵I VEGF, the amount of VEGF measured inthe scaffolds did not change significantly after day 2. The presentstudy limited the time frame for SBF incubation to 5 days, since thiswas a period in which mineral growth was initiated, and significantmineral growth occurred in a previous study on gas foamed/particulateleached 85:15 PLG scaffolds.

Previous studies on mineralized PLG scaffolds show that mineral growthcontinues for at least two weeks in vitro and it considered thatbioactive factors may incorporate into the mineral film for longerincubation periods. Notably, the attenuation of the release of VEGF fromPLG scaffolds caused by mineral formation cannot be explained byincorporation of the protein into the mineral film, since thisattenuation occurs primarily within the first 5 days of SBF incubation,and there is no significant incorporation during this time period.

Because the attenuation of growth factor release from PLG scaffoldscannot be explained by incorporation of proteins back into the scaffoldsafter release, or incorporation of proteins into the growing mineralcrystals, it is likely that the attenuation is simply due to a barriereffect. The mineral crystal growing on the inner pore surfaces of thePLG scaffold may physically block the release of proteins from thepolymer matrix. This barrier effect has been studied extensively incontrolled drug delivery applications using layered polymericmicrospheres and microspheres encapsulated in microporous membranes, andthe growth of bone-like mineral represents a new method for blockingprotein diffusion out of polymeric materials.

Thus, vascular endothelial growth factor does not incorporate into themineral film or significantly incorporate into the polymer scaffoldduring incubation of PLG in SBF. The previously observed effect ofmineral growth on VEGF release from PLG scaffolds is likely caused bythe mineral acting as a physical barrier to protein diffusion out of thescaffold. This mechanism is contemplated to be useful in controlled drugdelivery applications, as the release profile from these materials couldbe predictably controlled by mineral film thickness and density.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of certain preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and methods and in the steps or in the sequence ofsteps of the methods described herein without departing from theconcept, spirit and scope of the invention. More specifically, it willbe apparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

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What is claimed is:
 1. A method for generating a patterned surface on abiocompatible material, comprising irradiating at least a firstphotosensitive surface of a biocompatible material with pre-patternedelectromagnetic radiation in the form of constructively anddestructively interfering electromagnetic radiation, thereby generatinga pattern on said at least a first surface of said biocompatiblematerial.
 2. The method of claim 1, wherein said biocompatible materialis fabricated in an implantable device.
 3. The method of claim 1,wherein said pre-patterned radiation is constructively and destructivelyinterfering radiation generated by impinging monochromatic radiation ona diffractive optical element that converts said monochromatic radiationinto constructively and destructively interfering radiation.
 4. Themethod of claim 3, wherein said monochromatic radiation is generatedfrom a laser.
 5. The method of claim 3, wherein said monochromaticradiation is generated from a mercury bulb.
 6. The method of claim 3,wherein said monochromatic radiation is generated from anelectromagnetic radiation source in combination with a filter.
 7. Themethod of claim 3, wherein said diffractive optical element is adiffractive lens, a deflector/array generator, a hemispherical lenslet,a kinoform, a diffraction grating, a fresnel microlens or a phase-onlyhologram.
 8. The method of claim 3, wherein said diffractive opticalelement is fabricated from a transparent polymer or glass.
 9. The methodof claim 8, wherein said diffractive optical element is fabricated froma transparent polymer selected from the group consisting of apoly(propylene), poly(methyl methacrylate), poly(styrene), and a highdensity poly(ethylene).
 10. The method of claim 3, wherein saiddiffractive optical element is a diffraction grating fabricated frommetal on glass, metal on polymer or metal with transmission apertures.11. The method of claim 3, wherein said diffractive optical element isfabricated from fused silica or sapphire.
 12. The method of claim 1,wherein said photosensitive surface is prepared by applying aphotosensitive composition to at least a first surface of saidbiocompatible material.
 13. The method of claim 12, wherein saidphotosensitive composition comprises a combined effective amount of atleast a first photoinitiator and at least a first polymerizablecomponent.
 14. The method of claim 13, wherein said photosensitivecomposition comprises a polymerization-initiating amount of at least afirst UV-excitable photoinitiator.
 15. The method of claim 14, whereinsaid photosensitive composition comprises a polymerization-initiatingamount of at least a first UV-excitable photoinitiator selected from thegroup consisting of a benzoin derivative, benzil ketal,hydroxyalkylphenone, alpha-amino ketone, acylphosphine oxide,benzophenone derivative and a thioxanthone derivative.
 16. The method ofclaim 13, wherein said photosensitive composition comprises apolymerization-initiating amount of at least a first visiblelight-excitable photoinitiator.
 17. The method of claim 16, wherein saidphotosensitive composition comprises a polymerization-initiating amountof at least a first visible light-excitable photoinitiator selected fromthe group consisting of eosin, methylene blue, rose bengal,dialkylphenacylsulfonium butyltriphenylborate, a fluorinateddiaryltitanocene, a cyanine, a cyanine borate, a ketocoumarin and afluorone dye.
 18. The method of claim 16, wherein said photosensitivecomposition further comprises a co-initiating amount of at least a firstaccelerator.
 19. The method of claim 18, wherein said photosensitivecomposition further comprises a co-initiating amount of at least a firstaccelerator selected from the group consisting of a tertiary amine,peroxide, organotin compound, borate salt and an imidazole.
 20. Themethod of claim 13, wherein said photosensitive composition comprises aphotopolymerizable amount of at least a first monomeric, oligomeric orpolymeric polymerizable component.
 21. The method of claim 20, whereinsaid photosensitive composition comprises a photopolymerizable amount ofat least a first polymerizable monomer selected from the groupconsisting of an unsaturated fumaric polyester, maleic polyester,styrene, a multifunctional acrylate monomer, an epoxide or a vinylether.
 22. The method of claim 13, wherein said photosensitivecomposition comprises a combined effective amount of an eosinphotoinitiator, a poly(ethylene glycol) diacrylate polymerizablecomponent and a triethanolamine accelerator.
 23. The method of claim 1,wherein said pre-patterned radiation is applied to at least a firstsubstantially level surface of said biocompatible material.
 24. Themethod of claim 1, wherein said pre-patterned radiation is applied to atleast a first contoured surface of said biocompatible material.
 25. Themethod of claim 1, wherein the pattern generated comprises a patternwith a resolution of between about 1 μM and about 500 μM.
 26. The methodof claim 25, wherein the pattern generated comprises a pattern with aresolution of between about 1 μM and about 100 μM.
 27. The method ofclaim 26, wherein the pattern generated comprises a pattern with aresolution of between about 10 μM and about 100 μM.
 28. The method ofclaim 26, wherein the pattern generated comprises a pattern with aresolution of between about 1 μM and about 10 μM.
 29. The method ofclaim 26, wherein the pattern generated comprises a pattern with aresolution of between about 10 μM and about 20 μM.
 30. The method ofclaim 1, wherein said method is executed at a temperature compatible tomammalian biological systems.
 31. The method of claim 1, wherein saidbiocompatible material is maintained on a temperature-controlled supportduring said irradiation.
 32. The method of claim 1, wherein the patterngenerated comprises a pattern of polar oxygen groups on at least a firstsurface of said biocompatible material.
 33. The method of claim 1,wherein said biocompatible material is operatively associated with abiologically effective amount of at least a first mineral, bioactivesubstance or biological cell.
 34. The method of claim 33, wherein saidbiocompatible material is operatively associated with a biologicallyeffective amount of at least two minerals, bioactive substances orbiological cells.
 35. The method of claim 34, wherein said biocompatiblematerial is operatively associated with a biologically effective amountof a plurality of minerals, bioactive substances or biological cells.36. The method of claim 33, wherein said biocompatible material isoperatively associated with a biologically effective amount of at leasta first mineral.
 37. The method of claim 36, wherein said biocompatiblematerial is operatively associated with a biologically effective amountof calcium.
 38. The method of claim 33, wherein said biocompatiblematerial is operatively associated with a biologically effective amountof at least a first bioactive substance or bioactive drug.
 39. Themethod of claim 38, wherein said biocompatible material is operativelyassociated with a biologically effective amount of at least a firstmarker protein, transcription or elongation factor, cell cycle controlprotein, kinase, phosphatase, DNA repair protein, oncogene, tumorsuppressor, angiogenic protein, anti-angiogenic protein, cell surfacereceptor, accessory signaling molecule, transport protein, enzyme,anti-bacterial agent, anti-viral agent, antigen, immunogen,apoptosis-inducing agent, anti-apoptosis agent, cytotoxin, hormone,neurotransmitter, growth factor, hormone, neurotransmitter or growthfactor receptor, interferon, interleukin, chemokine, cytokine, colonystimulating factor, chemotactic factor, extracellular matrix componentor an adhesion molecule, ligand or peptide.
 40. The method of claim 39,wherein said biocompatible material is operatively associated with abiologically effective amount of growth hormone, parathyroid hormone(PTH), bone morphogenetic protein (BMP), transforming growth factor-α(TGF-α), TGF-β1, TGF-β2, fibroblast growth factor (FGF),granulocyte/macrophage colony stimulating factor (GMCSF), epidermalgrowth factor (EGF), platelet derived growth factor (PDGF), insulin-likegrowth factor (IGF), scatter factor/hepatocyte growth factor (HGF),fibrin, collagen, fibronectin, vitronectin, hyaluronic acid, anRGD-containing peptide or polypeptide, an angiopoietin or vascularendothelial cell growth factor (VEGF).
 41. The method of claim 38,wherein said biocompatible material is operatively associated with abiologically effective amount of at least a first bioactive DNAmolecule, RNA molecule, antisense nucleic acid, ribozyme, plasmid,expression vector, viral vector or recombinant virus.
 42. The method ofclaim 33, wherein said biocompatible material is operatively associatedwith a biologically effective amount of at least a first biologicalcell.
 43. The method of claim 42, wherein said biocompatible material isoperatively associated with a biologically effective amount of at leasta first bone progenitor cell, fibroblast or endothelial cell.
 44. Themethod of claim 43, wherein said biocompatible material is operativelyassociated with a biologically effective amount of at least a first boneprogenitor cell selected from the group consisting of a stem cell,macrophage, fibroblast, vascular cell, osteoblast, chondroblast andosteoclast.
 45. The method of claim 42, wherein said biocompatiblematerial is operatively associated with a biologically effective amountof at least a first recombinant cell that expresses at least a firstexogenous nucleic acid segment that produces a transcriptional ortranslated product in said cell.
 46. The method of claim 33, whereinsaid biocompatible material is operatively associated with a combinedbiologically effective amount of at least a first bioactive substanceand at least a first biological cell.
 47. The method of claim 41,wherein said biocompatible material is operatively associated with acombined biologically effective amount of at least a first osteotropicgrowth factor or osteotropic growth factor nucleic acid and a cellpopulation comprising bone progenitor cells.
 48. The method of claim 46,wherein said biocompatible material is operatively associated with acombined biologically effective amount of VEGF or a VEGF nucleic acidand a cell population comprising endothelial cells.
 49. The method ofclaim 33, wherein said at least a first mineral, bioactive substance orbiological cell is incorporated into said biocompatible material priorto the generation of said patterned surface.
 50. The method of claim 33,wherein said at least a first mineral, bioactive substance or biologicalcell is incorporated into said biocompatible material during orsubsequent to the generation of said patterned surface to form a patternof minerals, bioactive substances or biological cells on least a firstsurface of said biocompatible material.
 51. The method of claim 50,wherein said at least a first mineral, bioactive substance or biologicalcell is incorporated into said biocompatible material during thegeneration of said patterned surface.
 52. The method of claim 50,wherein said at least a first mineral, bioactive substance or biologicalcell is incorporated into said biocompatible material subsequent to thegeneration of said patterned surface.
 53. The method of claim 50,wherein at least a first mineral is incorporated into said biocompatiblematerial during or subsequent to the generation of said patternedsurface to form a mineralized biocompatible material comprising apattern of minerals on least a first surface.
 54. The method of claim53, wherein at least a first mineral is incorporated into saidbiocompatible material subsequent to the generation of said patternedsurface by exposure of said patterned surface to a mineral-containingsolution in vitro.
 55. The method of claim 53, wherein at least a firstmineral is incorporated into said biocompatible material subsequent tothe generation of said patterned surface by exposure of said patternedsurface to a mineral-containing body fluid in vivo.
 56. The method ofclaim 55, wherein at least a first mineral-adherent biological cell issubsequently bound to said mineralized biocompatible material to form apattern of biological cells on least a first surface of saidbiocompatible material.
 57. The method of claim 56, wherein said atleast a first mineral-adherent biological cell is bound to saidmineralized biocompatible material by exposure of said mineralizedbiocompatible material to a population of mineral-adherent cells invitro.
 58. The method of claim 56, wherein said at least a firstmineral-adherent biological cell is bound to said mineralizedbiocompatible material by exposure of said mineralized biocompatiblematerial to a population of mineral-adherent cells in vivo.
 59. Themethod of claim 1, wherein said biocompatible material comprises atleast a first portion that is a biodegradable material.
 60. The methodof claim 1, wherein said biocompatible material comprises at least afirst portion that is a non-biodegradable material.
 61. The method ofclaim 1, wherein said biocompatible material comprises at least a firstportion that is a substantially 2-dimensional biomaterial film.
 62. Themethod of claim 1, wherein said biocompatible material comprises atleast a first portion that is a 3-dimensional biomaterial scaffold. 63.The method of claim 1, wherein said biocompatible material comprises atleast a first portion that has an interconnected or open pore structure.64. The method of claim 1, wherein said biocompatible material comprisesat least a first portion that is fabricated from a metal, bioglass,aluminate, biomineral or bioceramic material.
 65. The method of claim64, wherein said biocompatible material comprises at least a firstportion that is fabricated from titanium or titanium coated with abiomineral.
 66. The method of claim 64, wherein said biocompatiblematerial comprises at least a first portion that is fabricated from abiomineral selected from the group consisting of hydroxyapatite,carbonated hydroxyapatite and calcium carbonate.
 67. The method of claim1, wherein said biocompatible material comprises at least a firstportion that is fabricated from a synthetic polymer or anaturally-occurring polymer.
 68. The method of claim 67, wherein saidbiocompatible material comprises at least a first portion that isfabricated from a synthetic polymer selected from the group consistingof a poly(vinyl alcohol), poly(ethylene glycol), pluronic,poly(vinylpyrollidone), hydroxyethyl cellulose, hydroxypropyl cellulose,carboxymethyl cellulose, poly(ethylene terephthalate), poly(anhydride)and poly(propylene fumarate).
 69. The method of claim 68, wherein saidbiocompatible material comprises at least a first portion that isfabricated from a polylactic acid (PLA) polymer, polyglycolic acid (PGA)polymer or polylactic-co-glycolic acid (PLG) copolymer.
 70. The methodof claim 67, wherein said biocompatible material comprises at least afirst portion that is fabricated from a naturally-occurring polymerselected from the group consisting of collagen, fibrin, matrigel,alginate, modified alginate, elastin, chitosan and gelatin.
 71. Themethod of claim 1, wherein said pre-patterned radiation isconstructively and destructively interfering radiation in the visiblespectrum.
 72. A method for forming a mineral pattern on a biocompatiblematerial, comprising preparing a patterned biocompatible material by theprocess of claim 1 and contacting said patterned biocompatible materialwith an effective amount of a mineral-containing solution.
 73. Themethod of claim 1, wherein said pre-patterned radiation isconstructively and destructively interfering radiation in the infraredspectrum.
 74. A 3-dimensional patterned biocompatible material preparedby the process of claim
 1. 75. The 3-dimensional patterned biocompatiblematerial of claim 74, further comprising a biologically effective amountof at least a first mineral, bioactive substance or biological cell. 76.The 3-dimensional patterned bicompatible material of claim 75, whereinsaid at least a first mineral, bioactive substance or biological cellforms a pattern on least a first surface of said biocompatible material.77. The 3-dimensional patterned biocompatible material of claim 76,comprising a biologically effective amount of at least a first mineralbound in a mineralized pattern to at least a first surface of saidbiocompatible material and a biologically effective amount of at least afirst biological cell bound to said mineralized pattern.
 78. Abiocompatible device comprising at least a first patterned 3-dimensionalportion prepared by the process of claim
 1. 79. The method of claim 1,wherein said pre-patterned radiation is constructively and destructivelyinterfering radiation in the ultraviolet (UV) spectrum.